A phase I clinical trial of replication-competent Ad4-HIVenv and Ad4-HIVmosaic gag vaccines has been initiated in the NIH clinical center in collaboration with NIAID and Dr. Mark Connors as Principal Investigator. The study is evaluating the safety and immunogenicity of the vaccines formulated as enteric coated capsules for oral administration and as a liquid for administration to the upper respiratory tract. This study follows a previous Phase I trial of an Ad4-flu vaccine, tested in the clinical center with Dr. Connors as PI. It's value for the HIV vaccine trial was to provide initial safety and dosage information, allowing jump-starting of the HIV vaccine trial. Data from the Ad4-flu study are now being evaluated. New Ad-recombinants are under development both for future clinical use and for pre-clinical studies in the rhesus macaque model. These include recombinants containing novel envelope inserts intended to generate broad, potent neutralizing antibodies. This work includes assessment of the immunogenicity of the newly constructed recombinant vaccines first in murine and subsequently in non-human primate models. In addition, Ad vectors with deletion of genes not essential for replication of the virus have been developed and expand the carrying capacity of the vaccine vector. They also are currently being evaluated in murine and non-human primate systems. One of the new recombinants is predicted to elicit enhanced immune responses. This is currently being evaluated in the rhesus macaque system. A new effort has been undertaken to allow sorting and characterization of HIV/SIV from clinical specimens using the new technology of nanoFACS. We have established the methodology to stain virions and sort them based on either cellular antigens incorporated into the virions or alternatively using specific antibodies to different envelope epitopes. The technique provides a sufficient yield of virions that allows downstream genomic and proteomic analysis.